Mammalian bombesin-like neuropeptides, gastrin releasing peptide (GRP) and neuromedin B (NMB), mediate a diverse range of biological responses in normal tissues, and stimulate the growth of some lung, pancreatic, and prostate carcinomas. Our laboratory cloned and characterized three structurally and pharmacologically distinct human bombesin receptors: the gastrin-releasing peptide receptor (GRP-R, or bb2); the neuromedin B receptor (NMB-R, or bb1); and bombesin receptor subtype 3 (BRS-3, or bb3). All three receptors are members of the G-protein coupled receptor superfamily, coupling to heterotrimeric G-proteins that activate phospholipase C. Several advances in our understanding of the function and regulation of these receptors has occurred within the last year: (1) we have identified four residues in the transmembrane domains of the receptor that are crucial for high-affinity binding of bombesin receptor agonists using site-directed mutagenesis; (2) we have used gene targeting strategies to create mice which lack the GRP-R, and progressed in our efforts to generate similar mice that lack NMB-R, or BRS-3. The phenotype of these mutants will be studied in the upcoming year; (3) we have developed a protocol that allows us to generate uncoupled receptors embedded in a normal biological membrane. This preparation allows us to reconstitute coupling to purified G-proteins, examining the selectivity and emzymology of receptor-catalyzed nucleotide exchange on heterotrimeric GTP-binding proteins. These studies show that the GRP-R couples selectively to the q subfamily of G-proteins, and not to Gi, Go, or transducin. This same preparation is also used to study receptor phosphorylation by G-protein receptor kinases in vitro, showing that GRK1 phosphorylates the GRP-R in a ligand dependent manner, with all the properties determined previously for GRP-R phosphorylation in vivo; (4) we are examining the GRP-R gene from patients with autism to determine if any allelic variants are associated with this disorder. The functional properties of these variants (ligand binding, coupling, internalization, sensitization, glycosylation, phosphorylation) will be examined in detail.